Inteins are naturally occurring, self-splicing protein subdomains that are capable of excising out their own protein subdomain from a larger protein structure while simultaneously joining the two formerly flanking peptide regions (“exteins”) together to form a mature host protein.
The ability of inteins to rearrange flanking peptide bonds, and retain activity when in fusion to proteins other than their native exteins, has led to a number of intein-based biotechnologies. These include various types of protein ligaton and activation applications, as well as protein labeling and tracing applications. An important application of inteins is in the production of purified recombinant proteins. In particular, inteins have the ability to impart self-cleaving activity to a number of conventional affinity and purification tags, and thus provide a major advance in the production of recombinant protein products for research, medical and other commercial applications.
Conventional purification tags provide a simple and robust means for purifying any tagged target protein, and are commonly added to desired target proteins through simple genetic fusions. These tags are now ubiquitous in research, and have formed a major platform for research and manufacturing of these important products. Once the tagged target protein is expressed in an appropriate host cell and purified via the tag, however, the presence of the tag on the purified target can lead to compromised activity, and potentially unwanted immunogenicity in the case of therapeutic protens. For these reasons, the ability to remove the affinity tag after purification is of critical importance in many applications, which is conventionally done through the addition of highly specific endopeptidase enzymes. Although these enzymes are generally effective, they are too expensive to scale up for manufacturing, and their use requires an additional step for their removal.
Thus, the ability of inteins to impart self-cleaving activity to conventional tags is a significant advance, and early implementations of intein-based self-cleaving affinity tag systems have been published in several patents and hundreds of journal papers in the biological sciences. Despite their strength, however, several substantial weaknesses remain that inhibit the full implementation of intein methods. In particular, the ability to tightly control the cleaving reaction in a variety of highly relevant contexts has been elusive. In order to be useful, the intein self-cleaving reaction must be tightly suppressed during protein expression and purification, but very rapid once the tagged target protein is pure. Of the two available classes of conventional inteins, one is highly controllable and is triggered to cleave by addition of thiol compounds, while the other is more loosely controlled and is triggered by small changes in pH and temperature.
Therefore, what is needed is a method for selective protein purification using a stable, transformative intein system. This system has significant utility in accelerated protein production and purification, with numerous applications in biological research, medicine and biopharmacueitical manufacturing. In particular, this intein system must be compatible with eukaryotic expression host systems, to be used for the expression and purification of complex glycoproteins. Some included areas of impact would be rapid anti-infectious disease vaccine manufacture, bioterrorism defense, and personalized anti-cancer antigen generation, as well as contributions to pure research and the acceleration of new drug evaluation and optimization.